Method for rapid and sensitive detection of IgM retroviral antibodies

ABSTRACT

A rapid and sensitive assay method for the detection of IgM antibody or the simultaneous detection of IgG and IgM antibody to retroviruses, including HIV-1 and HIV-2, and diagnostic test kits for carrying out the method is provided. According to the method of the invention, results are obtainable within 70 minutes.

TECHNICAL FIELD

The present invention relates to a rapid and sensitive assay method forthe detection of IgM antibody to retroviral antigens and thesimultaneous detection of IgM and IgG antibodies to retroviral antigens.The present invention also relates to test kits for carrying out theassay method.

BACKGROUND OF THE INVENTION

Assay systems capable of detecting the presence or absence of antibodiesgenerated in response to the presence of viral antigens are well known.Such assay systems have proved useful in, inter alia, the diagnosis ofvarious disease and infectious states, for example, acquired immunedeficiency syndrome (AIDS), AIDS related complexes (ARC or pre-AIDS),T-lymphocytic leukemia, and T-lymphocytic lymphoma.

The known assay systems, which employ antibody-antigen binding,ordinarily are designed to detect solely the presence or absence of IgG(immunoglobulin G). The appearance of detectable IgG directed toantigens in an infected/immunized individual, in many instances, doesnot occur until 30-40 days after initial infection. Typically, the IgGclass antibodies are often present for months or years after infectionor immunization with a foreign agent, such as a virus.

The presence of circulating IgG directed to an immunizing antigen duringthe course of infection or immunization is preceded by the presence ofcirculating IgM (immunoglobulin M) antibody directed against theantigen/immunogen. IgM antibody is often present as early as 14 days,possibly earlier, after infection/immunization. Unlike IgG antibodywhich remains for months or years after infection, IgM antibody losestiter 30-35 days after initial infection.

It is widely recognized that diagnostics which can detect antibodiesother than IgG are desirable. For example, it is known that generallyafter confrontation with a foreign agent, the human immune systemresponds by generating antibodies against the foreign agent or antigen.It is believed, as previously discussed, that IgM, not IgG, is producedfirst. IgM is, however, a relatively short-lived antibody. While it maybe produced shortly after infection, IgM specific antibodies fall,eventually below detectable levels, as IgG is produced in increasingamounts. Because IgM has a short life span, IgM specific antibodies aretypically below detectable levels before many diseases can even bediagnosed using known diagnostic assay methods.

As can be appreciated, assays capable of detecting IgM will be useful infacilitating early detection of various infections and diseases. Earlydiagnosis facilitates treatment, and minimizes the risk of spreadinginfection.

The present invention overcomes the limitations associated withconventional assay techniques because it provides a Quick Western Blotassay for detecting the presence or absence of IgM class antibody aloneor simultaneously with the detection of IgG.

Infections caused by the human retroviruses result in the appearance ofantibodies in the serum, and other body fluids of the infected victim.

AIDS, which was first diagnosed in 1981, is known to be caused by ahuman retrovirus called HIV-1 (Human Immunodeficiency Virus-1). It hasrecently been reported that another human retrovirus, designated HIV-2also causes AIDS.

The human retroviruses which are associated with AIDS are believed to betransmitted through intimate sexual contact as well as through blood.

Recently, investigators have become interested in two other humanretroviruses, designated HTLV-I and HTLV-II. (HTLV means HumanT-lymphotropic virus). Thus far, neither HTLV-I nor HTLV-II has beenidentified as the specific causative agent for any particular disease.These retroviruses, however, have been isolated from patients diagnosedwith T-lymphocytic leukemia and lymphoma. Antibodies to HTLV-I have beenalso found in patients diagnosed with chronic, progressive spasticparaparesis (PSP) secondary to myelopathy. Neurology Alert, Vol. 6, no.11, July 1988. It is presently believed that HTLV-I and HTLV-II aretransmitted through blood. As more information about these retrovirusesis revealed, it is predicted that as with HIV-1, all blood supplies willbe screened for the presence of antibodies to HIV-2, HTLV-I and HTLV-II.Thus, as previously mentioned, it is advantageous to be able to detectthe presence of IgM antibodies as well as IgG antibodies to facilitateearly detection of infections and minimize the risk of spreading suchinfection.

DISCLOSURE OF THE INVENTION

The present invention provides a rapid and sensitive test system fordetecting the presence of IgM antibody alone or simultaneously with IgGantibody against human retroviruses, including HIV-1, HIV-2, HTLV-I,HTLV-II and Equine Infectious Anemia Virus. The assay system of theinvention is a modified Quick Western Blot technique which comprisesincubating electrophoretically resolved viral protein with a testsample, preferably in the presence of nonfat milk proteins orpolyethyleneglycol (PEG) 8000.

It has been found that milk proteins and PEG act to both enhance andaccelerate specific protein binding. Consequently, test results areobtained in 70 minutes.

Like conventional Western Blot assays (such as those described by V. C.Tsang, J. Peralta, and R. Simmons, In: Methods in Enzymology, Vol. 92,Chapter 9, 1983, Academic Press, Inc.), the present method appliesblotting techniques but, according to the present method, about 20-40%more viral lysate is loaded onto each electrophoresis gel. Inparticular, from about 60-130 ug of viral lysate is loaded onto each10×16 cm gel rather than the 50-100 ug of viral protein used inconventional Western Blotting. However, it should be understood that theamount of viral lysate may be varied depending on the particular virus,for example, for HIV-1, HIV-2 and HTLV-II it is presently preferred toload the gels with from 60-120 ug of viral lysate, and most preferably70-80 ug. For HTLV-I it is presently preferred to use 60-130 ug of viralproteins and most preferably 80-100 ug. Concentrations of test sampleare also increased from 2-10 times over those used in the standardWestern Blot, preferably 5 times. In other words, in carrying out astandard Western Blot, test samples are typically diluted 1:100, whereasin the present method it is preferred to dilute the test sample fromabout 1:10-1:50 and most preferably 1:20. The increase in lysate andsample concentrations facilitates a drastic reduction in incubationtimes and thus, results are obtained in under 70 minutes.

It is known that the amount of protein subjected to electrophoresis inthe Western Blot assay is inversely related to the distinctiveness ofthe resulting bands, the potential for "noise bands" increasing with theamount of protein. Noise bands often appear adjacent to critical proteinbands and may be mistaken therefor, making evaluation of test resultsdifficult and increasing chances of false positives. It has,accordingly, previously been assumed that the use of relatively lowantigen lysate concentrations, i.e., from about 5-10 ug of protein/8×10cm gel (50-100 ug of protein/10×16 cm gel), are necessary to facilitateaccurate detection of antibodies to viruses. It has now been found (seeU.S. Pat. No. 4,816,387) that both antigen and sample concentrations canbe increased as previously described and the assay time drasticallydecreased as compared with the time required for conventional WesternBlot.

In one preferred embodiment of the present invention, a diagnostic testkit is provided which among other obvious advantages, such as timereduction, permits on site testing for antibodies, such as those againstthe AIDS virus. The diagnostic kit of the invention preferably includespredeveloped strong and weak positive and negative reference strips forevaluating the test results by visual comparison with test strips.However, to reduce costs, photographs of such strips may be substituted.Accordingly, reading the results of the test is facilitated. On sitetesting is particularly important in an organ transplant situationbecause testing can be performed on a 24 hour basis at virtually anylocation, and test results can be obtained within 70 minutes,comfortably within the life span of ischemic organs.

DETAILED DESCRIPTION OF THE INVENTION Resolution of the Viral Antigen

In accordance with the method of the invention, antigen concentrate iselectrophoretically resolved. HIV-1, HIV-2 and HTLV-I may be obtainedfrom Protatek International, St. Paul, Minn. The antigen concentrate isdiluted in buffer to a protein concentration at least 20%-40% greaterthan the 50-100 ug protein/10×16 cm gel utilized in conventional WesternBlot. Preferably, as previously mentioned, the antigen concentrate isdiluted in buffer to a protein concentration of about 60 to 120 ug per10×16 cm gel for HIV-1, HIV-2, and HTLV-II. Concentrations of 60 to 130ug per 10×16 cm gel are preferred for HTLV-I. The preferred dilutionbuffers are 0.05M TRIS-HCl/50% glycerol, pH 8, 2.5% SDS (sodium dodecylsulfate) and 5% mercaptoethanol or TRITON X100 in PBS buffer. Otherbuffers known to those skilled in the art are also suitable, such as 9Murea in 0.01M TRIS-HCl.

As noted above, the protein concentration of the antigen lysate usedwith the method of the invention is approximately 20 to as much as 40%higher than the 50-100 ug/10×16 cm gel typically used for conventionalWestern Blot. See pages 380 to 381 of the guidelines published by V. C.Tsang, J. Peralta, and R. Simons, in Methods Of Enzymology, Vol. 92,Chapter 29, 1983, Academic Press Inc., which sets forth the 5-10 ug/8×10cm gel and 50-100 ug/10×16 cm gel workable ranges of proteinconcentrations used in the conventional Western Blot assay. Beforeresolution, the antigen is generally first inactivated by means of knownpsoralen/UV irradiation techniques, and solubilized by treatment withfrom 0.1 to 1.0% TRITON X100 in PBS buffer. However, other inactivationand solubilization methods as are known to those skilled in the art mayalso be used. Then, the antigen is subjected to conventional gelelectrophoresis of the type reported by Tsang et al, Methods inEnzymology, Vol. 92 (1983).

A tracking dye is preferably added to the diluted antigen to producevisible protein banding. The preferred dye is bromophenol blue. The dyeis preferably prepared by dissolving 50 mg of bromophenol blue in 8 mlof glycerol, plus 1 ml each of 0.5M TRIS-HCl at pH 8.0 and H₂ O. Otherdyes, known to those skilled in the art, may also be used.

Suitable gels for the electrophoresis are also prepared in accordancewith the method of Tsang et al., Methods in Enzymology, Vol. 92 (1983).A 10% resolving polyacrylamide gel with a 3% stacking gel (SDS-PAGE) ispreferred because it resolves a molecular weight range of 12,000-180,000daltons, the molecular weight range embracing the proteins of HIV-1,HTLV-I and HIV-2. However, the percentage of polyacrylamide gel may varyfrom 8-11% depending upon the particular virus being resolved.

In accordance with a preferred embodiment of the present invention, theresolved antigen protein and test sample (including controls) areincubated in the presence of milk proteins, preferably defattedproteins. (It is believed that the presence of fat interferes with thetest.) Suitable defatted milk proteins include Carnation® lowfat milkpowder, but any defatted milk proteins as may be known are also useful.Such milk proteins are known to constitute about 60 to 90% casein, aphosphoprotein rich in serine; and from 10 to 40% of various otherproteins including lactoalbumin, lactoglobulin, membrane globulin and asmall amount of alkaline phosphatase, peroxidase catalase and xanthinedehydrogenase. The milk proteins may be-introduced into the assay systemin one of several ways.

According to the first embodiment of the invention, the nitrocellulosepaper blotted with the resolved viral antigen protein is coated with themilk proteins from a solution of buffer (e.g., PBS-Tween 20), containingabout 5 to 10% milk proteins for about 60 minutes. The paper is thenwashed in a buffer solution not containing milk proteins (e.g.,PBS-Tween 20) at room temperature for 1-5 minutes. The treated paper isthen stored under humid conditions until use.

According to a second embodiment of the invention, the milk proteins arethoroughly mixed with and dissolved in the buffer solutions containingthe test sample and controls. The controls and serum samples are thenincubated with the nitrocellulose strips for 15 to 20 minutes. Theliquid of each tube is discarded and the strips are washed with aPBS-Tween buffer at pH 7.3-7.4. The washing cycle consists of four 1minute washings.

According to a third embodiment of the invention, the milk proteins areprecoated on the nitrocellulose strips and additionally mixed with anddissolved in the buffer solution.

In accordance with another preferred embodiment, the resolved antigenprotein and test sample (including controls) are incubated in thepresence of polyethyleneglycol (PEG). Preferably, PEG is added to thesample dilution buffer in a concentration of from about 3 to 10% w/v andpreferably 5% w/v. It should be understood that both milk proteins andPEG can be used together to enhance antigen-antibody binding.

The nitrocellulose sheets are then cut into strips approximately 2-2.5mmin width. Each strip, after appropriate labelling, is placed in aseparate test tube for determination of antibodies to HIV-I viral lysateby the enzyme linked immunoassay of the invention. The nitrocellulosestrips can also be placed in incubation trays for in-house testing. Itshould be understood, however, that an uncut sheet can be placed in anincubation tray equipped with a pressing cover rather than cut intoindividual strips. This technique may be well suited to in-house asopposed to on-site testing. As can be appreciated, however, on-sitetesting is facilitated by use of individual tubes. Also, placing thestrips in individual tubes minimizes the need for handling during theassay procedure and thus, possible smearing of the fragile proteinpatterns with fingerprints. Using strips is also more economical thanuncut sheets because less reagent is necessary to carry out the test.

Test samples, strong and weak positive and negative references are addedto the tubes containing the nitrocellulose strips blotted with resolvedantigen. Test samples include, but are not limited to serum, semen andother body fluids. The positive reference is typically a sample known tocontain antibodies to the particular viral lysate. Positive referenceshave been obtained from the Centers for Disease Control (CDC), Atlanta,Georgia. Positive controls for HIV-2 and HTLV-I have been obtained fromGenetic Systems, Seattle, Washington. Alternatively, a positivereference may be made from any sample which has been standardized with apositive reference obtained from the CDC. Standardization typicallymeans that the same test results were obtained in about 20 repeatedruns. The positive reference is diluted 1/20 (1 part positive referenceto 20 parts buffer) in PBS (phosphate buffered solution)-tween pH(7.2-7.4), preferably containing 5% nonfat milk proteins. Typically, 150ul of the positive reference is mixed with 3 ml PBS-Tween.

The negative control is a sample known to be devoid of antibodies to theviral lysate, and is prepared by diluting 1/20 with PBS-Tween, pH7.2-7.4. Typically, 150 ul of a negative reference is mixed with 3 mlPBS-Tween. Negative references for HIV-1 have been obtained from theCDC. Negative references for HIV-2 and HTLV-I have been obtained fromGenetic Systems, Seattle, Washington.

As the assay method of the invention is a qualitative rather thanquantitative determination, the positive and negative references areused to evaluate the test results by comparison with the resultsobtained from test samples.

A reagent control may also be included as a quality control feature ofthe present invention and is used to assure accurate functioning of thetest. Normally, the reagent control is the buffer used to dilute testsamples and controls. Preferably, PBS-Tween, pH 7.2-7.4 is used as thereagent control. However, the reagent control is not necessary duringroutine readings of the strips.

As indicated hereinabove, when employing the milk protein treatmentand/or PEG of the present invention, test samples may be used which aremore concentrated than those used in conventional Western Blot toaccelerate the binding of antibody against the antigen contained in thestrips.

For serum samples, three to five times the concentration utilized in theconventional Western Blot assay is required, i.e., a dilution of onepart serum to twenty parts buffer as compared to the 1:100 dilutionfactor used in the Western Blot assay. (See Tsang et al., Method inEnzymology, Vol. 92, 1983.) Theoretically, the actual dilution factorfor particular samples may be varied, however, depending upon whether aspecimen gives an extremely weak positive response. A PBS-Tween, pH7.2-7.4 buffer is preferred for the dilution of samples. Preferably, thebuffer contains 5% nonfat milk and/or 5% w/v PEG 8000. Typically, 150 ulof a serum sample is mixed with 3 ml of the buffer. But other knownbuffers may be substituted.

The strips are then incubated with the positive and negative references,controls and test samples at room temperature, preferably for about 10to 20 minutes, to permit the binding of any antibodies to the viralantigen present in the sample to the antigen in the nitrocellulosestrips. A 15 minute incubation period is particularly preferred toinsure optimum binding of weak positives. However, if the assay isperformed without milk proteins or other binding enhancers such as PEG,a 20 minute incubation period is preferred.

Incubation with the Enzyme-Conjugated Antiserum

The liquid content of each tube is discarded, with the strips remainingin place in the tubes. The strips are then washed, preferably withPBS-Tween buffer at pH 7.2-7.4. In particular, the washing cycleincludes four 1 minute washings with PBS-Tween. The strips are thenincubated with an enzyme-conjugated anti-human IgM, alone or mixed withan enzyme-conjugated anti-human IgG for about 10 to 20 minutes, at roomtemperature, to permit binding of the enzyme conjugated antiserum orantisera to any antibody which bound to the antigen during the firstincubation period. Goat anti-human antisera-horseradish peroxidaseconjugate may be employed, although other enzyme conjugated antisera asare known to those skilled in the art may be used. For example, goatanti-human antisera conjugated with alkaline phosphatase may be used.Again, a 20 minute incubation period is preferred.

As can be appreciated if the assay is to be performed solely todetermine whether there is any antibody to the virus present in thesample, irrespective of the antibody class, i.e., IgG or IgM, then bothantisera may have the same label, i.e., HRP. Of course, if it isdesirable to differentiate between any IgG and IgM antibodies which maybe present, it is necessary to employ two different labels. Any labelsor combination as are known to those skilled in the art are suitable,for example, goat anti-human IgM labelled with alkaline phosphatase andgoat anti-human IgG labelled with horseradish peroxidase are suitable.Horseradish peroxide labelled goat anti-human IgG is available fromProtatek International, St. Paul, Minnesota; horseradish peroxidaselabelled goat anti-human IgM and alkaline phosphatase labelled goatanti-human IgM are available from Calbiochem as order Nos. 401905 and401902, respectively.

After completion of the incubation, the liquid content of each tube isdiscarded. The strips are then washed. Preferably the washing cycleincludes four 1 minute washings with PBS-Tween followed by one 1 minutewashing with either PBS or distilled water.

Incubation with an Enzyme Substrate

Then, the strips are incubated with one or more enzyme substrates (colorchange indicators) for about 10 minutes at room temperature, forproduction of at least one color. Substrate selection is dictated by theenzyme or enzymes used. One appropriate substrate for use withhorseradish peroxidase (HRP) enzyme is3,3'diaminobenzidinetetrahydrochloride dihydrate (DAB). DAB is availablefrom Aldrich Chemical Company, Inc., Milwaukee, Wisc. as Catalog No. 26,189-0. If as previously described, antisera labelled with differentenzymes are used, i.e., horseradish peroxidase and alkaline phosphatase(ALH) are used, then two different substrates should also be used. ThusDAB may be used to develop HRP producing a brown color and BCIP/NBT maybe used to develop ALH producing a purple color. BCIP(5-Bromo-4-chloro-3-indolyl phosphate-toluidine salt) is available fromBio-Rad as Catalog No. 170-6539 and NBT (p-Nitro blue tetrazoliumchloride) is available from Bio-Rad as Catalog no. 170-6532.

As can be appreciated, this results in a bi-color assay system fordetecting and distinguishing IgM and IgG antibodies which reacted withthe antigen on the test strip.

In practice, when two different labels are employed the strips arereacted with the substrates sequentially to develop the differentcolors. For example, in one preferred embodiment the strips are firstincubated for 10 minutes with DAB reactive with HRP to produce a browncolor. The first color producing reaction is stopped and the liquid isdiscarded and the strips washed 3 times for 1 minute each with a sodiumbicarbonate buffer to deactivate any remaining DAB and adjust the pH ofthe strips to a pH of approximately 9.2. Then, the strips are incubatedfor 10 minutes with BCIP/NBT reactive ALH to produce a purple color.BCIP and NBT are prepared and used in accordance with manufacturer'sinstructions.

It should be understood that the color producing reaction sequence maybe reversed so that the ALH is developed first. Moreover, and aspreviously stated, any known combination of enzymes and substrates maybe used. Also, it should be understood that one of the antisera may befluorescein conjugated rather than enzyme conjugated.

After the final incubation period, the second color producing reactionis stopped by addition of distilled water or 2N H₂ SO₄ and the resultsdetermined according to standard techniques such as those reported byTsang et al., Methods in Enzymology, Vol. 92 (1983).

In accordance with the present invention, determination of the presenceof retroviral antibodies can be accomplished in under 70 minutes. Itshould be understood that the present assay can also be performed in theabsence of milk proteins or PEG. If such assay conditions are selected,it is suggested that maximum incubation times, and maximum antigen-testsample concentrations be used to insure sufficient binding of anyantibody present to the antigen.

The Diagnostic Test Kit

In accordance with a preferred embodiment of the invention, aself-contained diagnostic test kit is provided which permits "on site"screening for antibodies to a variety of viruses, including HIV-1,HIV-2, HTLV-I, HTLV-II and Equine Infectious Anemia Virus. The test kitincludes a set of tubes containing strong and weak positive and negativereferences, and at least 1 buffer tube containing a predetermined volumeof buffer to which the test sample is added in a predetermined amount toobtain a dilution of from 1:10-1:50, preferably 1:20. A reagent controlmay also be included. The reference and control tubes are prediluted,and thus, the user need only dilute the test sample. A set of striptubes is also provided, each tube containing a nitrocellulose stripcontaining resolved antigen protein, electrotransferred from an SDS-PAGEgel loaded with from 60-130 ug of protein/10×16 cm gel to obtain aconcentration of antigen from 20% to 40% higher than that used inconventional Western Blot.

As indicated hereinabove, in a preferred embodiment a milk proteinadditive is either pre-dissolved in the buffer solutions containing thetest sample and the positive and negative references, or coated on thenitrocellulose strips containing the resolved antigen protein or both.Alternatively, the milk proteins may be separately provided in powderform to be added to the buffer by the user; the resulting buffersolution can then be directly mixed with the test and reference samples,or used to coat the test strips. As previously discussed, PEG may besubstituted for milk protein as a binding enhancer or if desired usedtogether with milk protein. Moreover, the assay can be performed in theabsence of either milk proteins or PEG.

In a preferred embodiment, the reference, control and sample tubes arenumbered. The strip tubes are assigned numbers corresponding to those onthe reference control and sample tubes. The strips are assigned numberscorresponding to the tubes in which they are placed. This type ofnumbering system avoids inadvertent mix-ups which can destroy theaccuracy of the assay. As can be appreciated, if the top of a tubecontaining a positive sample is placed on a tube containing a negativesample, it is likely to obtain a false positive result.

The kit also contains vials of enzyme-conjugated antisera reagents, atleast one substrate or color change indicator, washing buffers and atleast one solution for terminating the color reaction. Goat anti-humanIgG antiserum-horseradish peroxidase and goat anti-human IgMantiserum-alkaline phosphatase are presently preferred as the enzymeconjugated anti-serum reagents. The preferred reaction terminating agentand washing buffers are distilled H₂ O, and PBS Tween and PBS,respectively.

Preferably, pre-developed positive and negative reference strips andreagent control strips are provided in the kit. These controls areprepared in substantially the same manner as previously described exceptthat after developing, the strips are air dried. The predeveloped stripsare used to evaluate the test results by a visual comparison with thetest strips after completion of a color reaction. The reagent control,as noted, may be provided to assure the accurate functioning of thereagents. The predeveloped reference and control strips are asignificant feature of the present invention because they facilitatereading the assay results and practically eliminate the need for askilled technician to evaluate the results. Rather than includingpredeveloped control strips, reading of the assay results may befacilitated by including photographs of strong and weak positive andnegative references.

Also, as the kit is self-contained, no laboratory equipment is needed.The advantages of such a kit are apparent, as it facilitates screeningfor antibodies at any time and virtually at any place, including remotegeographic areas and those locations lacking a 24 hour testing facility.As aforementioned, this is of utmost importance in certain organtransplantation situations, in particular, if infection with the AIDSvirus is suspected.

The following specific examples of the assay method described hereinfurther illustrate the nature of the present invention, although it isunderstood that the invention is not limited thereto.

EXAMPLE 1 Detection of IgM Antibody Against HIV-1

70 ug HIV-1 antigen lysate was psoralen/UV inactivated, solubilized with1.0% TRITON X100 in PBS and electrophoretically resolved in themolecular weight of 12,000-160,000 daltons with a 10% 10×16 cmpolyacrylamide gel containing 3% SDS. The resolved antigen waselectro-transferred to nitrocellulose paper which was cut into strips. Adilution buffer of PBS-Tween containing 5% by weight Carnation® nonfatmilk was prepared. The HIV-1 strips were placed in trays in separatetroughs and 3 ml of test samples and controls diluted 1:20 (150 ul into3 ml buffer), were added to each strip and incubated on a rocker at roomtemperature for 15 minutes. After completion of the incubation periodthe liquid from each trough was removed and the strip washed four timesfor one minute each with a PBS-Tween wash buffer. Then, 3 ml ofantihuman IgM conjugated with HRP diluted 1:500 in PBS-Tween was addedto each strip and incubated on a rocker for 15 minutes at roomtemperature. After completion of the incubation, the liquid was removedand the strips washed four times for one minute with 3 ml of PBS-Tweenfollowed by a one-minute washing with 3 ml of PBS alone.

The strips were then incubated with 3 ml of DAB (12.5 mg DAB dissolvedin 25 ml of PBS containing 0.01% H₂ O₂) for 10 minutes at roomtemperature and covered with aluminum foil to produce color. The colorproducing reaction was stopped after 10 minutes by adding 3 ml ofdistilled water to each strip. The results were evaluated by visuallycomparing the developed test sample strips against the control strips.

EXAMPLE 2 Simultaneous Detection of IgM and IgG Antibody Against HIV-2

HIV-2 viral lysate was inactivated by psoralen/UV treatment andsolubilized with 1.0% TRITON X100 in PBS buffer. 85 ug of theinactivated, solubilized lysate were loaded onto a 10×16 cm 10%polyacrylamide gel containing 3% SDS and electrophoretically resolved inthe molecular weight range of 12,000-180,000 daltons. The resolved HIV-2antigen was electro-transferred onto nitrocellulose sheets which wasthen cut into strips.

The strips, placed into individual troughs on a tray, were incubated ona rocker at room temperature for 15 minutes with 3 ml of known HIV-2positive test samples (kindly provided by Genetic Systems) diluted 1:20in PBS-Tween buffer containing 5% by weight of nonfat milk proteins(Carnation® nonfat dry milk) and 5% PEG 8000). The diluent buffercontaining both milk protein and PEG is designated CB-Accel buffer.After completion of the incubation period, the liquid was removed andthe strips washed 4 times for one minute each with 3 ml of PBS-Tweenbuffer. The strips were then incubated on a rocker for 15 minutes atroom temperature with 3 ml of a mixture of antihuman IgM conjugated withAHL and antihuman IgG conjugated with HRP. Each antiserum was diluted1:500 in PBS-Tween buffer. After the incubation period, the liquid wasremoved and the strips were washed with 3 ml of PBS-Tween four times forone minute each followed by a one minute wash with 3 ml of distilledwater. Then 3 ml of DAB was (prepared as previously described inExample 1) was added to each strip and incubated for 10 minutes toproduce a first color. After completion of the incubation, the liquidwas removed and the strips washed twice with 3 ml of sodium bicarbonatebuffer at pH 9.2. Then 3 ml of BCIP/NBT in sodium bicarbonate buffercontaining 0.01% N.N.dimethylformamide was added to each strip andincubated for 10 minutes at room temperature. The strips were coveredwith aluminum foil. After completion of the incubation, the second colorproducing reaction was stopped by adding 3 ml of distilled water. Theresults were evaluated by visually comparing the developed test samplestrips with the control strips.

While preferred embodiments of the invention have been described, itwill be apparent to those of ordinary skill in the art that variouschanges and modifications may be made therein without departing from thespirit and scope of the invention. Accordingly, the above descriptionshould be construed as illustrative, and not in a limiting sense, thescope of the invention being defined by the following claims.

We claim:
 1. An assay method for detecting IgM antibodies to aretrovirus selected from the group consisting of HIV-1, HIV-2, HTLV-I,and HTLV-II, comprising the steps of, within 70 minutes:(a) contactingnitrocellulose paper containing blotted resolved retrovirus antigenprotein obtained from gel electrophorectically resolved viral lysatewith a test sample and incubating the nitrocellulose paper and testsample to permit binding of antibodies present in the sample to theprotein on the nitrocellulose paper wherein(1) the viral lysate has anantigen protein concentration at least 20% to 40% greater than the50-100 ug of antigen protein per 10×16 cm electrophoresis gel utilizedin the conventional Western Blot assay; and (2) the test sample isdiluted in buffer in a ratio of test sample to buffer of from 1:10 to1:50; (b) contacting the incubated nitrocellulose paper of step (a) withan anti-IgM enzyme conjugated antiserum reactive with said antibodies,and incubating to permit binding of the antiserum to said antibodies;(c) contacting the incubated nitrocellulose paper of step (b) with anenzyme substrate specific for the enzyme of step (b), and incubating tothereby produce color; (d) stopping the color producing reaction of step(c); and (e) evaluating the amount of color produced as an indication ofthe presence of antibodies to the viral lysate.
 2. The method of claim1, wherein the test sample is serum.
 3. The method of claim 1, whereinthe anti-IgM is conjugated with alkaline phosphatase or horseradishperoxidase.
 4. The method of claim 1, wherein step (a) is carried out inthe presence of from 3-10% nonfat milk protein.
 5. The method of claim4, wherein the milk protein is mixed with the buffer used to dilute saidtest sample.
 6. The method of claim 4, wherein said milk proteincomprises from 60 to 90% by weight casein, and from 10 to 40% by weightof a material selected from the group consisting of lactoglobulin,membrane globulin, alkaline phosphatase, peroxidase catalase, xanthinedehydrogenase, and mixtures thereof.
 7. The method of claim 4, whereinthe nitrocellulose paper containing the blotted resolved retrovirusantigen protein is coated with milk protein prior to incubation with thetest sample in step (a).
 8. The method of claim 1, wherein step (a) iscarried out in the presence of 3 to 10% polyethylene glycol.
 9. Themethod of claim 8, wherein the polyethylene glycol is mixed with thebuffer used to dilute said test sample.
 10. The method of claim 7,wherein the viral lysate has an antigen protein concentration of between60-130 ug per 10×16 cm gel.
 11. The method of claim 7, in which step (a)is repeated with at least one positive control (a sample containingantibodies to said retrovirus) and one negative control (a sample thatis devoid of antibodies to said retrovirus) in place of the test sample,the reaction product formed thereby is subjected to steps (b-e), and thecolors produced as compared as standards with the color produced fromthe test sample, to evaluate the presence of antibodies to theretrovirus in the test sample.
 12. An assay method for simultaneouslydetecting IgG and IgM antibodies to a retrovirus selected from the groupconsisting of HIV-1, HIV-2, HTLV-I, and HTLV-II, comprising the stepsof, within 70 minutes:(a) contacting nitrocellulose paper containingblotted resolved retrovirus antigen protein obtained from gelelectrophorectically resolved viral lysate with a test sample andincubating the nitrocellulose paper and test sample to permit binding ofantibodies present in the sample to the protein on the nitrocellulosepaper wherein(1) the viral lysate has an antigen protein concentrationat least 20% to 40% greater than the 50-100 ug of antigen protein per10×16 cm electrophoresis gel utilized in the conventional Western Blotassay; and (2) the test sample is diluted in buffer in a ratio of testsample to buffer of from 1:10 to 1:50; (b) contacting the incubatednitrocellulose paper of step (a) with an anti-IgM enzyme conjugatedantiserum reactive with said antibodies and with an anti-IgG enzymeconjugated antiserum reactive with said antibodies, and incubating topermit binding of said antisera to said antibodies; (c) contacting theincubated nitrocellulose paper of step (b) with at least one enzymesubstrate specific for the enzyme or enzymes of step (b), and incubatingto thereby produce color; (d) stopping the color producing reaction ofstep (c); and (e) evaluating the amount of color produced as anindication of the presence of IgM and IgG antibodies to the virallysate.
 13. The method of claim 12, wherein the test sample is serum.14. The method of claim 12, wherein the anti-IgM antiserum is conjugatedwith one enzyme and the anti-IgG antiserum is conjugated with adifferent enzyme.
 15. The method of claim 14, wherein the enzymes arehorseradish peroxidase and alkaline phosphatase.
 16. The method of claim12, wherein step (a) is carried out in the presence of from 3-10% nonfatmilk protein.
 17. The method of claim 16, wherein the milk protein ismixed with the buffer used to dilute said test sample.
 18. The method ofclaim 16, wherein said milk protein comprises from 60 to 90% by weightcasein, and from 10 to 40% by weight of a material selected from thegroup consisting of lactoglobulin, membrane globulin, alkalinephosphatase, peroxidase catalase, xanthine dehydrogenase, and mixturesthereof.
 19. The method of claim 16, wherein the nitrocellulose papercontaining the blotted resolved retrovirus antigen protein is coatedwith milk protein prior to incubation with the test sample in step (a).20. The method of claim 12, wherein step (a) is carried out in thepresence of from 3 to 10% polyethylene glycol.
 21. The method of claim20, wherein the polyethylene glycol is mixed with the buffer used todilute said test sample.
 22. The method of claim 12, wherein the virallysate has an antigen protein concentration of between 60-130 ug per10×16 cm gel.
 23. The method of claim 12, in which step (a) is repeatedwith at least one positive control (a sample containing antibodies tosaid retrovirus) and one negative control (a sample that is devoid ofantibodies to said retrovirus) in place of the test sample, the reactionproduct formed thereby is subjected to steps (b-e), and the colorsproduced are compared as standards with the color produced from the testsample, to evaluate the presence of antibodies to the retrovirus in thetest sample.
 24. A diagnostic test kit for simultaneously detecting,within 70 minutes, IgG and IgM antibodies which bind to an antigen of aretrovirus selected from the group consisting of HIV-1, HIV-2, HTLV-I,and HTLV-II, comprising:(a) a set of control tubes comprising positiveand negative references; (b) at least one reagent control tube; (c) atleast one dilution tube containing a predetermined volume of buffer fordilution of test samples in a ratio of test sample to buffer of from1:10 to 1:50; (d) a set of tubes containing nitrocellulose test stripscontaining resolved retroviral antigen, said antigen being obtained fromgel electrophorectically resolved viral lysate, wherein the resolvedretroviral antigen is present in a concentration at least 20% to 40%greater than the 50-100 ug of antigen protein per 10×16 cmelectrophoresis gel utilized in the conventional Western Blot assay; (e)an anti-IgG enzyme conjugated antiserum and an anti-IgM enzymeconjugated antiserum for reacting with said antibodies; (f) at least onecolor producing enzyme substrate specific for the enzyme or enzymes ofstep (e) wherein the color produced is an indicator to ascertain whetherthe antibodies are present; and (g) predeveloped positive and negativereference strips and reagent control strips for evaluating the resultsof the test by visually comparing the predeveloped strips with the teststrips after completion of a color change reaction.
 25. The diagnostictest kit of claim 24, wherein the dilution tubes contain 3-10% nonfatmilk protein dissolved in the buffer.
 26. The diagnostic test kit ofclaim 25, wherein said milk protein comprises from 60 to 90% by weightcasein, and from 10 to 40% by weight of a material selected from thegroup consisting of lactoglobulin, membrane globulin, alkalinephosphatase, peroxidase catalase, xanthine dehydrogenase, and mixturesthereof.
 27. The diagnostic test kit of claim 24, further comprising anonfat milk protein reagent for binding with the resolved retroviralantigen, said reagent being dissolved in the buffer in dilution tube (c)or coated on the nitro-cellulose test strips in tubes (d).
 28. Thediagnostic test kit of claim 24, wherein the dilution tubes contain fromabout 3 to 10% polyethylene glycol.
 29. The diagnostic test kit of claim28, wherein the polyethylene glycol is mixed with the buffer of step(c).
 30. The diagnostic test kit of claim 24, wherein the anti-IgMantiserum is conjugated with one enzyme and the anti-IgG antiserum isconjugated with a different enzyme.
 31. The diagnostic test kit of claim30, wherein the IgM antiserum is conjugated with alkaline phosphataseand the IgG antiserum is conjugated with horseradish peroxidase.
 32. Thediagnostic test kit of claim 24, wherein the electrophoreticallyresolved antigen has a concentration of 60 to 130 ug per 10×16 cm gel.33. A diagnostic test kit for simultaneously detecting, within 70minutes, IgM antibodies which bind to an antigen of a retrovirusselected from the group consisting of HIV-1, HIV-2, HTLV-I, and HTLV-II,comprising:(a) a set of control tubes comprising positive and negativereferences; (b) at least one reagent control tube; (c) at least onedilution tube containing a predetermined volume of buffer for dilutionof test samples in a ratio of test sample to buffer of from 1:10 to1:50; (d) a set of tubes containing nitrocellulose test stripscontaining resolved retroviral antigen, said antigen being obtained fromgel electrophoretically resolved viral lysate, wherein the resolvedretroviral antigen is present in a concentration at least 20% to 40%greater than the 50-100 ug of antigen protein per 10×16 cmelectrophoresis gel utilized in the conventional Western Blot assay; (e)an anti-IgM enzyme conjugated antiserum for reacting with saidantibodies; (f) at least one color producing enzyme substrate specificfor the enzyme of step (e) wherein the color produced is an indicator toascertain whether the antibodies are present; and (g) predevelopedpositive and negative reference strips and reagent control strips forevaluating the results of the test by visually comparing thepredeveloped strips with the test strips after completion of a colorchange reaction.
 34. The diagnostic test kit of claim 33, wherein thedilution tubes contain 3-10% nonfat milk protein dissolved in thebuffer.
 35. The diagnostic test kit of claim 33, further comprising anonfat milk protein reagent for binding with the resolved retroviralantigen, said reagent being dissolved in the buffer in dilution tube (c)or coated on the nitro-cellulose test strips in tubes (d).
 36. Thediagnostic test kit of claim 33, wherein the dilution tubes contain fromabout 3 to 10% polyethylene glycol.
 37. The diagnostic test kit of claim36, wherein the polyethylene glycol is mixed with the buffer of step(c).
 38. The diagnostic test kit of claim 33, wherein theelectrophoretically resolved antigen has a concentration of 60 to 130 ugper 10×16 cm gel.
 39. The diagnostic test kit of claim 33, wherein saidmilk protein comprises from 60 to 90% by weight casein, and from 10 to40% by weight of a material selected from the group consisting oflactoglobulin, membrane globulin, alkaline phosphatase, peroxidasecatalase, xanthine dehydrogenase, and mixtures thereof.